Biology Reference
In-Depth Information
The homology of the
gyrB
sequences between
V. parahaemolyticus
and
V. alginolyticus
is 86.8% (Venkateswaran et al
.,
1998). For this reason,
Venkateswaran et al. (1998) developed a PCR procedure using primers
VP-1/VP-2r (Table 5.4) targeting a 285-bp sequence of the
gyrB
gene for
specifi c detection of
V. parahaemolyticus
.
The
toxR
gene was fi rst discovered as the regulatory gene of the cholera
toxin operon and was later found to regulate many other genes in
V.
cholerae
(DiRita, 1992; Miller et al.,
1987). The
toxR
gene is well conserved
among species of
Vibrio.
The degree of homology of the
toxR gene
between
V. parahaemolyticus
and
V. cholerae
is 52% which is much lower than the
value of 91-92% for the rRNA gene (Kita-Tsukamoto et al
.,
1993; Lin et al.,
1993). Based on these earlier observations, Kim et al
.
(1999) developed a
DNA colony hybridization test with the use of a 678-bp polynucleotide
probe (Lin et al., 1993) for the
toxR
gene of
V. parahaemolyticus
, to confi rm
the identity of isolates. Kim et al. (1999) also developed a specifi c PCR
assay for the identifi cation of
V. parahaemolyticus
based on amplifying
amplicons of the
toxR
gene. Three effective primer pairs were identifi ed
(
Table 5.2
). These primer sequences were selected from the regions of the
toxR
gene not conserved between
V. parahaemolyticus
and
V. cholerae
(Lin
et al. 1993). A total of 373 strains of
V. parahaemolyticus
were all found to
carry the
toxR
gene.
Blackstone et al. (2003) developed a Rti-PCR assay for detection of
V. parahaemolyticus
in oysters with the use of a pair of primers amplifying
labeled fl uorogenic probe. Their procedure involved homogenizing oyster
tissue at a 1:10 dilution in alkaline peptone water (pH 8.5) followed by
overnight enrichment incubation at 35°C. The assay detected target DNA
from 1 CFU per PCR reaction.
A PCR method for quantitative detection of
Vibrio parahaemolyticus
was
developed by Wang and Levin (2004). The primers
L-tdh/ R-tdh (Table
2) from Bej et al
.
(1999) were used. Several lysis methods were compared
and a lysis solution designated TZ developed by Abolmaaty et al. (2000)
proved effective.
Molecular Typing of
V
.
parahaemolyticus
Wong et al. (1996) found that the restriction nuclease
Sfi
I yielded 17 clear
and discernible PFGE bands with 130 clinical strains of
V. parahaemolyticus
from Thailand resulting in a total of 39 discernible PFGE patterns.
Wong et al. (1999) subjected 308 clinical isolates of
V. parahaemolyticus
derived from food outbreaks in Taiwan between 1993 and 1995 to RAPD
generated 41 RAPD patterns. The patterns were grouped into 16 RAPD
